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Molecular cloning of a DNA-end-binding protein (Ku antigen) recognized by autoantibodies and its application.

自身抗体识别的DNA末端结合蛋白（Ku抗原）的分子克隆及其应用。

基本信息

批准号：
62570296
负责人：
MIMORI Tsuneyo
金额：
$ 1.34万
依托单位：
Keio University School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1987
资助国家：
日本
起止时间：
1987 至 1988
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-62570296/
关键词：
Autoimmune Disease Autoantibody Ku antigen Molecular cloning cDNA RFLP ELISA 自己免疫患抗Ku抗原

项目摘要

Anti-ku autoantibodies in patients with PSS-PM overlap syndrome recognize a 70kD/80kD protein heterodimer which binds to ends of dsDNA. In this project, we isolated and characterized cDNA clones that encode the Ku autoantigen, and intended their clinical application.In the first year, a human hepatoma cell cDNA library inserted into the EcoRl site of lambda gtll phages were screened with an anti-Ku serum to identify plaques expressing Ku epitopes. Three positive clones (K14, K68 and K71) were isolated and deomnstrated to express fusion proteins recognized by anti-Ku antibodies. In the second year, Okayama-berg cDNA library was screened by using a previously isolated cDNA encoding the partial 80kD sequence (K71) as a probe for colony hybridization. The clone Ku80-6 that contained the longest cDNA insert (3.4kb, ldentical with the larger mRNA from two mRNAs hybridized by K71) was isolated, and its nucleotide sequence was determined by Sanger's dideoxy method. The Ku80-6 cDNA contained a … More single long open reading frame encoding 732 amino acids (Mr=82,713) followed by 1082 bases of 3'-non-coding region and a poly(A) tail. The sequence of the Ku80-6 was compared with previously described sequences using the computer search, but no significant homology was found either in DNA (GenBank) or protein (NBRF) data bank. In Southern blot, the Ku80-6 hybridized with 4-5 DNA bands from human leukocyte DNA digested with various restriction enzymes. It was noted that RFLP was seen in the 3.4kb-HindIII fragment and likely to associate with SLE patients who had anti-ULRNP antibodies. Using purified fusion proteins expressing from K68(70kD) and K71(80kD) clones, we developed ELISA to detect anti-Ku antibodies. When sera from various collagen diseases were screened by this assay, anti-Ku antibodies were mostly detected in patients with overlap syndrome, consisted with the result of a conventional immunodiffusion assay.Our cDNA encoding the Ku antigenic proteins will be a powerful tool to study not only the structure and function of the Ku antoantigen but also pathogenetic mechanisms of autoimmune diseases. Less

PSS-PM重叠综合征患者的抗ku自身抗体识别与dsDNA末端结合的70 kD/80 kD蛋白异源二聚体。本项目分离并鉴定了编码Ku自身抗原的cDNA克隆，并计划将其用于临床应用。第一年，用抗Ku血清筛选插入λ gtll噬菌体EcoRl位点的人肝癌细胞cDNA文库，以鉴定表达Ku表位的噬斑。分离到3个阳性克隆（K14、K68和K71），经鉴定，它们表达了抗Ku抗体识别的融合蛋白。第二年，以已分离的80 kD部分序列（K71）为探针，对Okayama-berg cDNA文库进行了筛选。克隆Ku 80 -6含有最长的cDNA插入片段（3.4kb，与K71杂交的两个mRNA中较大的mRNA相同），用桑格双脱氧法测定其核苷酸序列。Ku 80 -6 cDNA含有一个 ...更多信息单个长开放阅读框编码732个氨基酸（Mr= 82，713），随后是1082个碱基的3 '-非编码区和一个poly（A）尾。将Ku 80 -6的序列与已报道的序列进行了计算机检索，但在DNA（GenBank）和蛋白质（NBRF）数据库中均未发现明显的同源性。在Southern杂交中，Ku 80 -6与经各种限制性内切酶消化的人白细胞DNA的4-5条DNA带杂交。结果表明，3.4kb-HindIII片段存在RFLP，且可能与SLE患者抗ULRNP抗体有关。用纯化的K68（70 kD）和K71（80 kD）融合蛋白建立了检测抗Ku抗体的ELISA方法。用该方法筛选各种胶原病患者血清时，大多数重叠综合征患者血清中均检出抗Ku抗体，与常规免疫扩散法的结果一致，我们获得的编码Ku抗原蛋白的cDNA将为研究Ku自身抗原的结构和功能以及自身免疫性疾病的发病机制提供有力的工具。少